Role of estradiol-17beta on nuclear and cytoplasmic maturation of pig oocytes.

The role of estradiol-17beta on nuclear and cytoplasmic maturation of pig oocytes was investigated in the present study. To determine the estradiol effect, oocytes were cultured for 42 h in a steroid free medium composed of mTCM-199 supplemented with LH, FSH and 10% charcoal extracted follicular fluid. Estradiol receptor (ER), detected by a binding assay, were present in cumulus cells and oocytes during maturation with higher levels observed at 24 h of culture in the oocytes and at 36 h in the cumulus cells. To block estradiol action an antiestrogen (1-p-dimethylaminoethoxyphenyl-1,2-diphenyl-1-butene (tamoxifen)) was added to the maturation medium at various concentrations. The percentage of treated oocytes that underwent nuclear maturation was similar (P>0.05) to the control group. Cytoplasmic maturation, determined by the ability to form female pronucleus (FPN) and male pronucleus (MPN), was not different (P>0.05) among all groups. The presence of 4-hydroxy-4-androstene-3-17-dione (4-OHA) also did not influence nuclear (P>0.05) or cytoplasmic maturation (P>0.05). The results suggest that estradiol is not involved in maturation of pig oocytes. However, the present experiment used pronuclei formation as the endpoint, no studies were done in regard to estradiol's effects on the embryonic development.

Preovulatory LH profiles of superovulated cows and progesterone concentrations at embryo recovery.

Forty-two Holstein cows were randomly assigned to three superovulatory treatment groups of 14 cows each. Cows in Group I received follicle stimulating hormone (FSH; 50 mg i.m.); those in Group II received FSH (50. mg i.m.) along with GnRH (250 ug in 2 % carboxymethylcellulose s.c.) on the day of estrus; and cows in Group III were infused FSH (49 mg) via osmotic pump implants. FSH was administered over a 5-d period for cows in Groups I and II (twice daily in declining doses). Cows in Group III received FSH over a 7-d period (constantly at a rate of 7 mg/day). All cows received 25 mg PGF(2)alpha (prostaglandin F(2)alpha) 48 hours after initiation of the FSH treatment. Blood samples were collected from seven cows from each group at 2 hour intervals on the fifth day of superovulation for serum luteinizing hormone (LH) concentration analysis by radioimmunoassay, and blood samples were collected from all cows on the day of embryo recovery for plasma progesterone determination. The LH profile was not altered (P>0.05) by either GnRH administration or by the constant infusion of FSH as compared to FSH treatment alone. Plasma progesterone concentrations were highly correlated with the number of corpora lutea (CL) palpated (r=0.92; P<0.01) and with the number of ova and/or embryos recovered (r=0.88; P<0.01). The accuracy of predicting the number of recoverable ova and/or embryos by the concentration of plasma progesterone was 86%.

Caries on root surfaces exposed following gingivectomy in conventional rats infected with Streptococcus sobrinus and Actinomyces viscosus.

To study the ability of bacteria associated with coronal caries to initiate root surface caries, a rat model was used. Root surfaces were exposed by gingivectomy in rats fed a caries-promoting diet and orally inoculated with either Actinomyces viscosus M-100, Streptococcus sobrinus (mutans) 6715, or both. A fourth group received a diet containing antibiotics. The animals were sacrificed 64 days following the gingivectomy performed on the right molar quadrants. Gingivectomy significantly increased exposed lingual root length and root caries incidence. There were no caries on non-gingivectomy root surfaces. Root surface caries incidence in the groups inoculated with A. viscosus and A. viscosus plus S. sobrinus did not differ. For both these groups, root caries incidence was significantly greater than that for the group inoculated with S. sobrinus alone. Root caries incidence in this latter group did not differ from that in the control group.

Influence of calcium on the true acrosome reaction and capacity of bull spermatozoa to penetrate oocytes in vitro.

A series of experiments were conducted to determine the role of Ca in several physiological functions of bovine spermatozoa. For spermatozoa incubated in the absence of Ca for up to 24 h, motility was not different from those incubated with Ca. For spermatozoa incubated in the continuous presence of Ca, true acrosome reaction values were 0% at 0 h, 1.5% at 6 h, and 6.0% at 12 h. Spermatozoa incubated in vitro for up to 12 h in the absence of Ca did not undergo a true acrosome reaction; however, when Ca was added during incubation, a synchronous true acrosome reaction was induced within 10 min (0% at 0 h, 8.5% at 6 h, and 8.5% at 12 h). When spermatozoa were preincubated in the presence or absence of Ca for 6 h, then added to zona-intact dead bovine oocytes and incubation continued with and without Ca for 18 h, the number of spermatozoa binding to and penetrating each oocyte was greater when Ca was present. Also, the percentage of oocytes being penetrated was greater when Ca was present. These results indicate that: 1) Ca is not necessary for maintenance of spermatozoan motility; 2) Ca is required for the induction of a true acrosome reaction among a population of spermatozoa; 3) Ca is able to induce the synchronous true acrosome reaction in a low percentage of spermatozoa; and 4) Ca is important in spermatozoan binding and initiation of penetration of oocytes.

Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species.

A simple dual stain procedure (DS) for simultaneously determining sperm viability and acrosomal status is described. The DS includes the use of vital stain trypan blue to detect live and dead spermatozoa and Giemsa to detect the presence or absence of an acrosome. For staining, spermatozoa are washed, incubated with trypan blue, washed, dried onto slides, and subjected to Giemsa. Dead spermatozoa stain blue in the postacrosomal region while live spermatozoa remain unstained. The acrosome stains light purple-dark pink while acrosome-free sperm remain unstained. This staining pattern enables differentiation of spermatozoa which have undergone a true acrosome reaction (TAR) from those which have undergone a false acrosome reaction (FAR). Incubation of bull, boar, ram, and stallion spermatozoa for 60 minutes at 37 degrees C in the presence of calcium ionophore A23187 increased the proportion of spermatozoa undergoing a TAR in all species except the stallion. Incubation of bull spermatozoa for up to 24 hours at 37 degrees C resulted in a decrease over time in the percentage of live acrosome-intact spermatozoa and a simultaneous increase in the percentage of spermatozoa categorized as having undergone a TAR and FAR. The DS could be a useful technique in evaluating sperm viability and acrosomal status in fertilization and clinical studies.

The effects of different levels of dietary sucrose on root caries subsequent to gingivectomy in conventional rats infected with Actinomyces viscosus M-100.

Three groups of weanling, Sprague-Dawley-derived rats were inoculated with Actinomyces viscosus M-100 and fed powdered diet containing either 67%, 5%, or 0% confectioner's sugar. Two further groups were fed diet containing 5% confectioner's sugar and inoculated with Streptococcus sobrinus 6715 or S. sobrinus 6715 plus A. viscosus M-100. The most coronal 1 mm of gingiva was removed from maxillary and mandibular right molar quadrants (gingivectomy), and the animals re-inoculated following gingivectomy. The animals were killed 64 days following gingivectomy, and the lingual surface of mandibular first molar roots was measured for exposed root-surface area and root caries. In the groups of rats infected with A. viscosus M-100, root caries area was significantly greater in the group fed diet containing 67% confectioner's sugar. Sucrose level did not significantly affect the amount of exposed lingual first molar root area regardless of whether the tooth had been subjected to a gingivectomy. In the groups of rats receiving diet containing 5% confectioner's sugar, there were no significant differences in root caries area or exposed root-surface area, regardless of the infection status of the animals. In the rat model presented here, a high level of dietary sucrose was a necessary condition for the initiation of root caries in the absence of other readily fermentable carbohydrates.

Viability of mouse half-embryos in vitro and in vivo.

Mouse embryos at the 2-, 4-, 8-cell, and morula stage were divided in half by using microsurgical procedures and were either grown in vitro up to the blastocyst stage or transferred at the late morula stage into the uteri of pseudopregnant recipients. A relatively high percentage of the half embryos from 2-cell (70%), 4-cell (75%), 8-cell (93%), or morula stage embryos (75%) developed into blastocysts in vitro. However, the overall development in vivo of half embryos was low, as 3%, 13%, 8%, and 1% of half embryos from the 2-cell, 4-cell, 8-cell, and morula stages, respectively, developed into live fetuses. Embryos which were divided in half at different stages developed at different rates in vitro. This determined the stage of embryonic development at the time of transfer, which might have interacted with the stage of pseudopregnancy of the recipients to influence embryo survival in vivo.

Effect of in vitro storage of bovine spermatozoa on acrosomal integrity, proteolytic activity, binding, and initiation of penetration of oocytes.

During a 10-day 5 degrees C storage and subsequent 4-6-hr 37 degrees C incubation, both percent live spermatozoa and percent spermatozoa with an intact acrosome decreased, and percent spermatozoa with a late-reacted or without an acrosome increased. When stored spermatozoa were mixed with oocytes, no decrease in percent of oocytes with spermatozoa bound or percent of oocytes with spermatozoa starting to penetrate occurred as storage time increased. A 58% decrease in acrosin gelatinolytic activity and a 56% decrease in acrosin esterolytic activity but no decrease in nonacrosin proteolytic activity were evident over the 10-day storage. These studies show that a change in acrosomal morphology as well as a loss of acrosin may be responsible for the decreased fertility following extended in vitro storage of mammalian spermatozoa.

A method of semi-quantitative microradiographic analysis of root surface lesion remineralization.

The purpose of this study was to present a modified photometer based digital analysis system for image enhanced microradiographic semi-quantitation of reactions of demineralization and remineralization of root surface lesions. The system was used to determine effects of fluoride on abraded root surface dentin in vitro. Lesions of 30-40 micron depth were produced during 18 h exposure of 180 micron thick single sections in solution of 3 mM Ca (Ca/P 1.67) and 30 mM lactic acid at pH 5.1. Mean mineral content of the lesions was 32% relative to adjacent sound dentin. Exposure of 20 h in the same acid with 5 mM Ca (Ca/P 1.67) and 0.12 mM NaF at pH 6.1 restored the mean mineral content to 76% of normal and increased the resistance of the lesions to demineralization. Remineralization and subsequent demineralization characteristics indicated the growth of fluorapatites on residual minerals in the dentin lesions. The microradiographic analysis system proved to be sensitive and critical in these studies.

Evaluation of the effects of F in acidified gelatin gel on root surface lesion development in vitro.

Oral exposed, natural root surfaces of extracted human teeth were subjected to artificial lesion formation in gels of dialyzed gelatin that contained 75 mM lactic and 25 mM acetic acid and 1, 3, 10, 32, 100, 320, 550, or 1,000 mg F/l at pH 4.3-4.4 and at 36 degrees C. Ca and PO4 demineralization per square millimeter exposed surface and lesion depth decreased with increased F concentrations, except for the levels above 320 mg/l. Quantitative microradiographic image analyses showed that the mean percent mineral content of the entire lesions relative to the adjacent sound tissue was constant at about 64% for the F levels less than 320 mg/l. F above 3 mg/l changed the mineral density profile of the lesions by eliminating subsurface lamination. This showed that sufficient F incorporated at the site of demineralization prevented lesion progression and that the best effect would be obtained for F levels between 100 and 320 mg/l. Higher levels of F may also prove to be beneficial by providing labile F from CaF2 formed in the root surface.

Comparison of two scoring methods for root surface caries in hamsters.

The aim of this investigation was to compare two methods of scoring root surface caries in hamster teeth. The experimental material was mandibles from hamsters which had been infected with Actinomyces viscosus, fed a caries-promoting diet, and received either 0, 1, 10, or 25 ppm fluoride in the drinking water during an 18-week experimental period (Stookey, 1986). Root surface caries on the lingual surfaces of mandibular first molars was scored according to the grid method of Doff et al. (1977) and by the quantitative planimetric method of Firestone et al. (1986). Data from both methods confirmed that 25 and 10 ppm fluoride were significantly more caries-inhibitory than 1 ppm fluoride, which, in turn, significantly reduced root surface caries compared with the 0 ppm control group. Correlation coefficients for caries scores (0.92) and ranking of the scores (0.88) for each animal between the two methods were highly significant (p < 0.0001). The planimetric measurement of root surface caries in hamsters was as sensitive as the grid-scoring method.

Immunoglobulins in piglets from sows heat-stressed prepartum.

Sows were subjected to moderate heat stress in a chamber (32 C) from d 100 of pregnancy until less than 8 h before delivery of first piglet, while control sows were in a thermoneutral chamber (21 C) or farrowing house (22 C). Blood serum and colostrum at parturition of heat-stressed sows and their piglets' serum at birth had elevated cortisol concentrations. Total protein, globulin and immunoglobulin G (IgG) concentrations in sow serum tended to decrease as parturition time was approached; albumin did not change. Total protein and IgG concentrations in colostrum at parturition and in milk 24 and 48 h later tended to be lower in heat-stressed sows. Concentrations of these four protein fractions (total, globulin, IgG and albumin) in piglet serum at birth did not differ among treatment groups, but soon after colostrum ingestion they increased markedly in all groups. Therefore, in all groups total protein remained constant while globulin and IgG decreased. Globulin concentration on d 1 was lowest in piglets from heat-stressed sows, but its rate of decrease after d 1 was not affected by sow treatment. Immunoglobulin G concentration was 11 mg/ml lower, but its rate of decrease through postnatal d 20 was slower in piglets from heat-stressed sows than in those from control sows; a 10-mg/ml difference in IgG concentration on postnatal d 1 has been associated with increased preweaning mortality in piglets. Higher cortisol concentration in serum and lower IgG in colostrum of sows under heat stress was associated in their piglets with higher serum cortisol at birth and lower serum IgG for the first 20 d postnatum.

In vivo capacitation and acrosome reaction of bovine sperm in estrous and diestrous cows.

A staining procedure which enables distinction between spermatozoa possessing a true and false acrosome reaction (AR) was utilized to assess the incidence of capacitation and the true AR of bull spermatozoa recovered from the uterine horns of estrous and diestrous cows. Results show that at 3 and 6 h post-insemination, approximately 14.5 and 31.5%, respectively, of the live spermatozoa recovered had undergone a true AR in the uterus of estrous cows. An increasing percentage of those spermatozoa recovered from estrous cows with time were categorized as undergoing a false AR. This suggests that spermatozoa underwent capacitation, a true AR, then died prior to fixation and staining, therefore being grouped as false acrosome-reacted. Few spermatozoa were observed to have undergone a true AR in diestrous cows. It is apparent from this study that individual spermatozoa undergo capacitation and a true AR at different times during incubation in utero in estrous cows.

Effects of GnRH treatment on superovulatory responses of dairy cows.

Twenty-eight Holstein cows were randomly divided into two groups. Group received 50 mg of FSH-P intramuscularly in declining doses for five days. Cows in group II received the same treatment as those in Group I but were also administered 250 ug of GnRH on the day of expected estrus. Neither the mean number of corpora lutea palpated nor the mean number of embryos recovered nonsurgically was different (P0.05) between treatments. However, administration of GnRH resulted in a higher fertilization rate and recovery of more developing embryos.

Nuclear decondensation of mammalian spermatozoa: changes during maturation and in vitro storage.

Swelling of the spermatozoan nucleus and decondensation of the chromatim occur soon after penetration of spermatozoa into the egg cytoplasm. This decondensation was duplicated in vitro by incubating pre-ejaculatory ram, rabbit and bovine spermatozoa and also stored post-ejaculatory bovine spermatozoa in 1% sodium dodecyl sulfate and 2.0 mM dithiothreitol. Spermatozoa obtained from the testis and epididymal caput, corpus and cauda showed a progressive resistance to nuclear decondensation, while no change was evident in the decondensation time of spermatozoa obtained from the epididymal cauda, vas deferens and ejaculated semen. There was also a significant increase in decondensation time after the spermatozoa had been stored in vitro at 25 degrees C. This increased resistance to nuclear decondensation in the in vitro stored spermatozoa, reflecting an increase in cross-linking within the spermatozoan histones by formation of disulfide bonds, may account for part of the increased embryonic mortality observed when spermatozoa are stored in vitro prior to insemination.